The stimulation of rat adrenal fasciculate cells by ACTH involves activation of cholesterol side-chain cleavage by a complex array of lipid and protein factors. Experiments are proposed that aim to resolve contributions to the stimulation of cholesterol metabolism by two proteins, a cycloheximide-sensitive peptide that is elevated by ACTH (sterol regulatory peptide (SRP)) and a sterol carrier protein (SCP2) that is insensitive to ACTH and cycloheximide. The possible involvement of A poprotein E and of fatty acid binding protein will also be examined. The contributions to stimulation from 5-hydroperoxy eicosatetroaenoic acid (5-HPETE) and from polyunsaturated acidic phospholipids, both of which are elevated by ACTH and directly stimulate steroidogenesis, will be investigated. Emphasis will be placed on the mechanism of action of SRP on intermembrane cholesterol transfer within adrenal mitochondria and the role of these other adrenal constituents in facilitating this process. Changes in mitochondria produced by the cellular action of ACTH (mediated by SRP) and by the direct action of SRP will be characterized with the aim of understanding this process. The presence of comparable mechanisms in the stimulation of cholesterol metabolism in primary culture of bovine adrenal cells will be evaluated. Several new techniques will be developed to study the actions of these mediators in virable cells and intact mitochondria. These include transfer of specific proteins and antibodies into cells, colloidl-gold immunohistochemistry of cells and mitochondria to localize SRP, SCP2, and P-450scc, and spectrophotometric methods for locating reactive mitochondrial cholesterol (using filipin and exogenous P-450scc). The membrane disposition of cytochrome P-450scc will be probed with a set of monoclonal antibodies raised against the cytochrome. The enhancement of cholesterol metabolism by polyunsaturated phospholipids in vesicles reconstituted with purified P-450scc will be compared by this and other techniques with the stimulatory effect of these phospholipids in intact mitochrondia. These experiments address several new aspects of intracellular cholesterol movement and metabolism that are central to steriod synthesis in all steroidogenic tissues.